Blue light-inducible system for gene expression

ABSTRACT

The present invention provides methods for light-dependent gene regulation using a light-responsive DNA-binding protein. Also provided are related nucleic acid molecules, and protein molecules, such as those encoding or comprising the light-responsive DNA-binding protein or DNA-binding sites recognizing the light-responsive DNA-binding protein. Kits using the present light-dependent gene regulation system are further provided by the present invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/359,030, filed May 16, 2014, which is a §371 national stage entry ofPCT/US2012/065493, filed Nov. 16, 2012, which claims priority of U.S.Provisional Application No. 61/561,585, filed Nov. 18, 2011, the entiredisclosure of which is incorporated herein by reference.

GOVERNMENT INTEREST

This invention was made with United States government support undergrant number R01 GM081875 awarded by the National Institutes of Health.The United States government has certain rights in the invention.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“UTSW004WO_ST25.txt”, which is 44 kilobytes as measured in MicrosoftWindows operating system and was created on Nov. 15, 2012, is filedelectronically herewith and incorporated herein by reference.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention relates generally to the field of molecularbiology. More specifically, the invention relates to methods andcompositions regarding light-responsive proteins and nucleic acids forgene expression.

II. Related Art

The ability to artificially control gene expression in eukaryotic cellsis essential for many applications in basic molecular biology, includingcell biology, biochemical or biomedical research. Most currentlyavailable gene regulatory systems are based on chemical inducermolecules (e.g. tetracycline) that must enter a cell to bind a targetprotein and activate its transcriptional activity. Such systemsgenerally have downsides that include the need for the addition of asmall chemical inducer and typically an inability to turn geneexpression on and off rapidly.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a recombinant nucleic acidmolecule comprising a sequence encoding a light responsive DNA bindingprotein (LRDP) comprising: a) a LOV domain; and b) a DNA binding domain(DBD), wherein said LOV domain and DBD are from a homologous species,and wherein said sequence encoding said LRDP is operatively linked to apolynucleotide encoding a heterologous transcriptional activationdomain. In one embodiment, the LRDP is an EL222-LOV LRDP.

In another aspect, the invention provides a polypeptide encoded by anucleic acid molecule of the present invention, for instance comprisinga sequence encoding a LRDP, wherein the LRDP comprises a LOV domain anda DNA binding domain from a homologous species operatively linked to apolynucleotide encoding a heterologous transcription activation domain.In a particular aspect, the invention provides a cell comprising such apolypeptide.

In yet another aspect, the invention provides a recombinant nucleic acidmolecule comprising an EL222-binding consensus sequence operably linkedto a heterologous transcribable polynucleotide sequence. In oneembodiment, the DNA binding site comprises a sequence selected from thegroup consisting of SEQ ID NOs: 4-66. In yet another embodiment, thetranscribable polynucleotide sequence operably linked to theEL222-binding consensus sequence is selected from the group consistingof a reporter sequence, a cell stress tolerance sequence, an industrialenzyme encoding sequence, a sequence encoding a biofuel productionenzyme, a sequence encoding a cell lysis protein, and a sequenceencoding a cell regulatory protein.

In still another aspect, the invention provides a cell comprising arecombinant nucleic acid molecule of the invention comprising anEL222-binding consensus sequence operably linked to a heterologoustranscribable polynucleotide.

Also provided by the invention is a cell or multicellular organismcomprising a nucleic acid molecule of the invention comprising a LRDPencoding sequence comprising a LOV domain and a DNA binding domain froma homologous species operatively linked to a polynucleotide encoding aheterologous transcription activation domain. In one embodiment, such acell or multicellular organism of the invention further comprises arecombinant nucleic acid molecule comprising an EL222 binding consensussequence operably linked to a heterologous transcribable polynucleotidesequence. In another embodiment, a cell or multicellular organism of theinvention may also comprise a second nucleic acid molecule comprising aDNA binding site for said DNA binding domain operably linked to aheterologous transcribable polynucleotide sequence. In certainembodiments, a cell or multicellular organism of the present inventionmay be selected from the group consisting of a bacterial cell, yeastcell, animal cell, mammalian cell, insect cell, fungal cell and plantcell.

In a particular aspect, the invention provides a method of activatingtranscription in a cell comprising illuminating a cell of the inventionwith blue light. In one embodiment, transcription in the cell isactivated by at least 10×, 11×, 12×, 13×, 14×, 15×, 16×, 17×, 18×, 19×,20×, 21×, 22×, 23×, 24×, 25×, 30×, 35×, 40×, 45×, 50×, 55×, 60×, 65×,70×, 75×, 80×, 85×, 90×, 95×, 100×, 105× or 110× relative totranscription in the absence of blue light. In another aspect, theinvention provides a method of inactivating blue-light inducedtranscription comprising diminishing blue light exposure to a cell ofthe invention.

In one aspect, the present invention provides a method of identifyinghigh-affinity DNA binding sites for a light responsive DNA bindingprotein (LRDP) comprising a LOV domain and a DNA binding domain (DBD),wherein said LOV domain and DBD are from a homologous species, whereinsaid method comprises: a) incubating a polypeptide comprising a LOVdomain and said DNA binding domain with a plurality of differentpolynucleotide sequences in the presence of blue light; b) removing saidblue light; and c) identifying polynucleotide sequences bound to saidDNA binding domain in the presence of blue light and released from saidDNA binding domain in the absence of blue light, wherein saididentifying comprises isolating and sequencing said polynucleotidesequences bound to said DNA binding domain in the presence of blue lightand released from said DNA binding domain in the absence of blue light,whereby a high-affinity DNA binding site is identified. In oneembodiment, the plurality of different polynucleotide sequences aredistributed on an array and said identifying comprises determining theidentity of said polynucleotide sequences bound to said DNA bindingdomain in the presence of blue light and released from said DNA bindingdomain in the absence of blue light on said array. In anotherembodiment, the polypeptide is immobilized on a surface. In yet anotherembodiment, the LRDP used in the above method comprises at least oneLRDP variant and/or may be operably linked to a detection moiety. Suchdetection moieties may be selected from the group consisting of isotopiclabel and optically detectable label, such as a fluorescent protein,green fluorescent protein variants or enzymatic labels. In anotherembodiment, the LRDP comprises a sequence encoding an EL222 LRDP variantcomprising altered blue light responsiveness as compared to wild-typeEL222 LRDP.

In another aspect, the invention provides a method of recruitingproteins to a surface in a light dependent manner comprising: a) coatingthe surface with DNA-binding sequence molecules; b) exposing the surfaceto a light responsive DNA binding protein (LRDP); and c) exposing thesurface to blue light. In one embodiment, the LRDP of the above methodmay be linked to a second polypeptide. Said method may optionallyfurther comprise the step of d) detecting the second polypeptide. Incertain embodiments, the second polypeptide may be linked to a detectionmoiety, for instance, an isotopic label or optically detectable label,such as a green fluorescent protein, green fluorescent protein variantsand enzymatic labels. In another embodiment, the DNA-binding sequencemolecules may comprise distinct variant sequences and/or may be coatedonto the surface at discrete locations. In yet another embodiment, theLRDP of the above method comprises at least one LRDP variant.

In yet another aspect, the present invention provides a method of lightdependent isolation of target moieties comprising: a) inserting into thegenome of cell a DNA binding site for a LRDP; b) extracting said genomefrom said cell or progeny thereof; c) applying said extracted genome toimmobilized LRDP in the presence of blue light, whereby said targetmoieties are isolated. In one embodiment, the target moieties of such amethod are selected from the group consisting of genomic fragments andDNA binding proteins.

In a still another embodiment, the invention provides a method ofaltering expression of a polynucleotide in a subpopulation of cellscomprising illuminating with blue light a discrete number of cells ofthe invention, for instance cells comprising a nucleotide of theinvention comprising a LRDP encoding sequence comprising a LOV domainand a DNA binding domain from a homologous species operatively linked toa polynucleotide encoding a heterologous transcription activationdomain, whereby the discrete number of cells exhibit altered geneexpression. In one embodiment, the method further comprises ceasing saidilluminating, whereby said expression reverts to baseline.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1A AND 1B. Show a model for the activation of the E. litoralis 222amino acid protein (EL222) by blue light and the light-responsiveEL222-based gene expression system. FIG. 1A shows a schematic overviewof EL222 architecture and signaling mechanism (Nash et al., 2011). FIG.1B demonstrates modification of EL222 for use in eukaryotic cells byadding a nuclear localization sequence (NLS) and a transcriptionalactivation domain (VP16-AD) from the herpes simplex virus VP16 protein.High affinity DNA binding sites (assembled in tandem repeats upstream ofa minimal promoter) were also identified. In the dark, the VP16-EL222chimera cannot activate a luciferase reporter construct under thecontrol of five-tandem copies of EL222 DNA binding sites (EL222-DBS);exposure to blue light activates the VP16-EL222 protein allowing it toturn on luciferase transcription.

FIGS. 2A AND 2B. Demonstrate the use of an in vitrooligonucleotide-protein binding selection assay (SELEX) (Tuerk and Gold,Science 249 (1990): 505) to identify high-affinity EL222 targetsequences. FIG. 2A shows a schematic of the SELEX enrichment protocol toidentify EL222 binding sites. FIG. 2B shows EMSA results of EL222binding to ³²P-labeled DNA pools derived from SELEX (or the previouslyidentified EL222 substrate AN-45; Nash et al., 2011); mixtures wereexposed to light for 25 min before loading and electrophoresing on anative PAGE gel.

FIGS. 3A AND 3B. Demonstrate mapping of the EL222 binding site withinthe SELEX-derived Clone-1 DNA. FIG. 3A shows a schematic of the scanninganalysis of Clone-1 (SEQ ID NO:68), which contains the 33-bp randomizedregion (upper case) flanked by 6 bp of primer binding sequence (lowercase). The locations of each 20 bp Clone-1-derived DNAs (SEQ IDNOs:63-66) are given for each fragment. FIG. 3B shows EMSA of EL222binding to each DNA, using the same conditions as FIG. 2A.

FIGS. 4A AND 4B. Demonstrate light-inducible transgene expression inHEK293T cells using VP16-EL222. FIG. 4A shows expression results for aFirefly luciferase reporter construct under the control of 5 copies of45 bp Clone-1 sequence (SEQ ID NO:68) co-transfected with the VP16-EL222expression construct into HEK293T cells. FIG. 4B shows expressionresults for a Firefly luciferase reporter construct under the control of5 copies of 20-bp C1-2 (SEQ ID NO:69) sequence similarly co-transfectedwith the VP16-EL222 expression construct into HEK293T cells. One daypost-transfection, the cells were kept in the dark or illuminated withblue light for 24 hr. Afterwards, cells were harvested and luciferaseactivity was measured (left graph). Luciferase values were normalizedfor transfection variability using a control Renilla luciferase reporterwith a constitutively-active CMV promoter and was co-transfected withthe Firefly luciferase reporter and VP16-EL222 expression vector. Thechange in transcription is expressed as a fold change (FC) inactivation, whereFC=[(Firefly/Renilla)_(VP16-EL222)/(Firefly/Renilla)_(empty vector)].The regulatory DNA sequence for each reporter construct is shown, the EL222-DBS is represented in upper case letters and the linker regionconnecting each EL222-DBS is represented in lower case letters. The 20bp region that corresponds to the C1-2 sequence is shown in bold. Dataare shown as the mean and the standard error from n=2 (pClone1-45[Luc])or n=3 (pClone1-20[Luc]) independent experiments each done intriplicate.

FIG. 5. Demonstrates improved effects on gene expression in HEK293Tcells using illumination protocols optimized for the lifetime of thewildtype EL222 photoexcited state using an illumination schedule of (20s light:60 s dark=20 s ON, 60 s OFF) through a 24 hr period. The figureshows expression results for a Firefly luciferase reporter constructunder the control of 5 copies of 20-bp C1-2 (SEQ ID NO:69) sequenceco-transfected with the VP16-EL222 expression construct into HEK293Tcells. The change in transcription is expressed as a fold change (FC) inactivation, whereFC=[(Firefly/Renilla)_(vP16-EL222)/(Firefly/Renilla)_(empty vector)].Data are shown as the mean and the standard error from n=3 independentexperiments each done in triplicate.

FIG. 6. Shows the nucleotide sequence encoding the NLS-VP16-EL222(14-222) (SEQ ID NO:70). Nucleotides 7-27, shown in italics andunderlined, corresponds to the region encoding the nuclear localizationsignal (NLS); nucleotides 28-261, shown in bold, correspond to theregion encoding VP16; and nucleotides 268-879, shown as boxed,correspond to the region encoding the EL222 (14-222) LOV-DNA bindingdomain.

FIGS. 7A-1, 7A-2, 7B-1 and 7B-2. Show that proper transcriptionalactivation in HEK293T cells requires both high affinity DNA binding siteand blue light illumination. FIGS. 7A-1 and 7A-2 demonstrate DNA bindingsite specificity, showing results from cells that were co-transfectedwith VP16 (empty) or VP16-EL222 and a reporter construct containingeither 5 copies of the 20-bp C1-2 sequence (SEQ ID NO: 69), 3 copies ofthe AN-45 sequence or 5 copies of the upstream activation sequence fromthe Gal4 gene (UAS_(G)). Cells were kept in the dark or illuminated withblue light pulses (20 s ON 60 s OFF; 2 W/m²) for 24 hr. FIGS. 7B-1 and7B-2 demonstrate that illumination with red light does not result intranscriptional activation.

FIG. 8. Shows that illumination with blue light to activate the lightresponsive DNA binding protein (LRDP) has no significant negative impacton HEK293T cell viability.

FIGS. 9A AND 9B-1, 9B-2 and 9B-3. Show visualization ofVP16-EL222-driven mCherry fluorescence in HEK293T cells. FIG. 9A showscells that were transiently transfected with vectors to co-express VP16(empty) or VP16-E222 and an mCherry reporter under the control of 5copies of the 20 bp C2-1 sequence (SEQ ID NO: 69). Cells were kept inthe dark or illuminated with blue light pulses (20 s ON, 60 s OFF; 2W/m²) for 24 hr prior to imaging on fluorescence microscope (10×magnification; 3 s exposure). FIGS. 9B-1, 9B-2 and 9B-3 show surfaceplot representations (done using ImageJ) of the results described inFIG. 9A.

FIGS. 10A, 10B AND 10C. Demonstrate that active site variants of EL222extend the lifetime of the light-state adduct. FIG. 10A shows mutationsin EL222 that alter the rate of activation of the protein localized tothe active site as indicated in the crystal structure for EL222 (Nash etal., PNAS 108 (2011): 9449). FIG. 10B demonstrates that EL222 variantstune the lifetime of the adduct state relative to WT (top line, τ˜30 s).V41I:V121I (third line from the top) and A79Q (second line from the top)both increase the lifetime of the adduct state 10-fold. Combining thesemutations with a further V52I variation increases the lifetime to amaximum of τ˜2000 s in a V41I/V52I/A79Q/V121I (AQTrip, bottom line)variant. The rate of dark-state recovery of the proteins was determinedby fitting the UV-visible absorbance spectra of EL222 at 450 nm afterillumination to first-order exponential curves (Zoltowski B D andGardner K H; unpublished results). FIG. 10C shows the calculated foldchange in activation (see FIG. 4) for VP16 fusion proteins of the A79Q,L52I, L52I/A79Q variants, made and co-transfected with the 5×20 bp C2-1(SEQ ID NO: 69) Luciferase reporter construct into HEK293T cells.

FIGS. 11A, 11B AND 11C. Show results from a cell line that stablyexpresses VP16-EL222 and shows higher reporter activation. FIG. 11Ashows Luciferase activity for Clone-3 and HEK293T cells that weretransfected with a vector containing 20 bp C2-1 sequence (SEQ ID NO: 69)only (see FIG. 5) and illuminated with blue-light pulses (20 s ON, 60 sOFF) or kept in the dark for 3, 6, 9, 12 and 24 hr before cells wereharvested. FIG. 11B also shows Luciferase activity as in FIG. 11A exceptcells were illuminated with blue-light pulses of 20 s ON 60 s OFF, 10 sON 70 s OFF, or 5 s ON, 75 s OFF, for 12 hr post-transfection. FIG. 11Cshows western blot results for Clone-3 (+VP16-EL222) and HEK293T(−VP16-EL222) cells transfected with the 5×20 bp C2-1 (SEQ ID NO: 69)Luciferase reporter and illuminated with blue-light pulses (10 s ON, 70s OFF) for 12 hr or kept in the dark. Cells lysates were made fromuntransfected cells (unt) and cells from each condition (+ or−VP16-EL222), these were blotted with antibodies against FireflyLuciferase or β-actin (loading control). The asterisk indicates anon-specific band detected by the anti-Luciferase antibody.

FIGS. 12A AND 12B-1, 12B-2 and 12B-3. Show visualization ofVP16-EL222-driven mCherry fluorescence in cells stably expressingVP16-EL222. FIG. 12A shows cells transfected with an mCherry reporterunder the control of either 5 copies of the 20 bp C2-1 sequence (SEQ IDNO: 69) or 5 copies of the UAS_(G) sequence as control. Cells were keptin the dark or illuminated with blue light pulses (20 s ON, 60 s OFF; 2W/m²) for 24 hr prior to imaging on fluorescence microscope (10×magnification; 3 s exposure). FIGS. 12B-1, 12B-2 and 12B-3 show surfaceplot representations (done using ImageJ) of the results described forFIG. 12A.

FIGS. 13A AND 13B. Demonstrate transcriptional activation by VP16-EL222is reversible and repeatable. FIG. 13A shows results for Clone-3 cellsthat were transfected with the 5×20 bp C2-1 (SEQ ID NO: 69) Luciferasereporter and either incubated in the dark for the entire experiment(black line) or illuminated with blue-light pulses (20 s ON, 60 s OFF)for two separate 3-hour periods (grey line). FIG. 13B shows normalizedvalues, for which the luciferase intensity values obtained for thedark-state condition samples were subtracted from the light-statecondition values.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO:1—The single-stranded oligonucleotide used for the SELEXprocedure.

SEQ ID NO:2—The forward L1 primer used to amplify DNA pool for SELEXprocedure.

SEQ ID NO:3—The reverse L1 primer used to amplify DNA pool for SELEXprocedure.

SEQ ID NO:4-SEQ ID NO:10—Seven DNA-binding site sequences recognizingthe EL222 DNA-binding domain identified through the SELEX procedure andchosen for follow up studies.

SEQ ID NO:11-SEQ ID NO:62—The remaining DNA-binding site sequencesrecognizing the EL222 DNA-binding domain identified through the SELEXprocedure.

SEQ ID NO:63-SEQ ID NO:66—Four overlapping 20 bp Clone-1 derivedsequences for further study (C1-1 through C1-4).

SEQ ID NO:67—The consensus EL222 binding motif sequence.

SEQ ID NO:68—The 45 bp pClone1-45 sequence comprising the 33-bprandomized region flanked by 6 bp of primer binding sequence.

SEQ ID NO:69—The 20-bp pClone1-20 sequence comprising the Clone-1derived C1-2 sequence and linker sequence.

SEQ ID NO:70—The nucleotide sequence encoding the VP16-NLS-EL222 fusionprotein.

SEQ ID NO:71—The VP16-NLS-EL222 fusion protein sequence.

SEQ ID NO:72—The nucleotide sequence encoding the 13-residue N-terminaltruncation of WT-EL222.

SEQ ID NO:73—The 13-residue N-terminal truncation of WT-EL222 amino acidsequence.

SEQ ID NO:74—The EL222 V41I variant amino acid sequence.

SEQ ID NO:75—The EL222 L52I variant amino acid sequence.

SEQ ID NO:76—The EL222 A79Q variant amino acid sequence.

SEQ ID NO:77—The EL222 A79R variant amino acid sequence.

SEQ ID NO:78—The EL222 A79T variant amino acid sequence.

SEQ ID NO:79—The EL222 V121I variant amino acid sequence.

SEQ ID NO:80—The EL222 V41I:L52I variant amino acid sequence.

SEQ ID NO:81—The EL222 V41I:A79Q variant amino acid sequence.

SEQ ID NO:82—The EL222 V41I:V121I variant amino acid sequence.

SEQ ID NO:83—The EL222 L52I:A79Q variant amino acid sequence.

SEQ ID NO:84—The EL222 V41I:L52I:V121I variant amino acid sequence.

SEQ ID NO:85—The EL222 V41I:L52I:A79Q:V121I variant amino acid sequence.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention provides a system for selectively and efficientlyactivating gene expression in a light-responsive manner. The presentdisclosure therefore provides methods, nucleic acid molecules, proteins,kits and assays related to light-dependent regulation of geneexpression. The present invention provides in certain embodiments alight-responsive DNA-binding protein operably linked to atranscriptional activation domain that functions to bind DNA andactivate transcription in response to blue light. In one embodiment, thelight-responsive DNA-binding protein is a prokaryotic protein, such as abacterial protein. In a specific embodiment, the protein is EL222 (E.litoralis 222 amino acid protein; Nash et al., Proc. Natl. Acad. Sci.108 (2011): 9449; incorporated herein by reference). In anotherembodiment, the present invention provides DNA-binding sites capable ofbeing recognized by a light-responsive DNA-binding protein of theinvention. In a particular embodiment, such DNA-binding sites may bedouble stranded and thus DNA-binding sites represented as singlestranded sequences herein may additionally comprise the complementthereof.

The present disclosure provides unique advantages over presentlyavailable gene expression systems. For instance, expression of only asingle protein is sufficient in the system to directly activatetranscription of a target gene in response to blue light. Thelight-responsive DNA-binding protein (LRDP) can be provided containingboth a light-oxygen-voltage (LOV) domain and DNA-binding domain.Regulation of the DNA-binding ability of the light-responsiveDNA-binding protein of the present invention can therefore be carriedout within the same molecule as the DNA-binding protein, in other words,in cis, which can yield higher fold activation as compared to thepresently available gene expression systems, such an at least 108-foldor greater up-regulation demonstrated in the Examples below.

In another embodiment, a light-responsive DNA-binding protein usedherein is comprised as a single molecule without the need toartificially create a protein comprising both the light-responsivedomain and the DNA-binding domain, such as a fusion protein. Thelight-responsive DNA-binding protein can then be operatively linked to aheterologous regulatory domain, such as one or more heterologoustranscriptional activation domains. To the contrary, currentphotosensitive gene expression systems are designed as light-regulated“two-hybrid” type systems that associate separate DNA binding and geneactivation components in a light-dependent manner.

The currently available light-dependent expression systems compriseadditional limitations or complications overcome by the presentinvention. In particular, several light-dependent expression systemsutilize a photosensitive protein requiring an unusual chromophore thatmust be exogenously provided for many different types ofbiologically-relevant host cells. The expression system of the presentinvention, however, relies on a commonly-available chromophore, flavinmononucleotide (FMN), eliminating the need to supply cells withexogenous small molecule precursors and/or engineer the expression ofenzymes to promote the formation of such chromophores. Furthermore, thelight-dependent expression systems currently available in the art arederived from eukaryotic sources increasing the potential for crosstalkand/or pleiotropic effects if utilized in hosts similar to theoriginating source. This crosstalk significantly limits the possibilityof successful use in many biologically-relevant host cells, such aseukaryotic host cells. The light-responsive DNA-binding proteins of thepresent system, however, are uniquely derived from a lower organism,such as a prokaryotic organism, and thus may be effectively used forgene expression in cells of higher organisms, such as eukaryotic cells.

In certain embodiments, the gene expression system of the presentinvention uses blue light (400-480 nm) to trigger gene expression. Theuse of light as a control signal provides several advantages, as it canserve as a noninvasive, nontoxic, selective and rapid inducer.Importantly, many cells and tissues are not photoresponsive andtherefore light is an ideal stimulus as it will not affect thephysiology of most cell lines used in research. In addition, lightallows for more precise spatial and temporal control of gene expressionbecause it can be easily turned-on/off and be directed at a specificregion or site in a cell, cell culture, tissue or organism. A key aspectof temporal control of such activation is that the photochemicalsignaling mechanism within a LOV domain shuts off quickly afterillumination ceases as a photochemically-generated bond spontaneouslycleaves. The rate of this cleavage can be modified using point mutationsto residues near the chromophore binding site. In one embodiment, thepresent invention provides LOV domain sequences with such pointmutations, for instance in the EL222 LOV domain. Thus, the use of lightas a stimulus to control gene expression in vivo avoids nearly all ofthe drawbacks attributed to the inducing chemical or treatment ofcurrently available systems.

In particular, most currently available gene regulatory systems arebased on chemical inducer molecules (e.g. tetracycline) that must entera cell to bind a target protein and activate its transcriptionalactivity. While these chemical-based systems are in widespread use, theyhave several key limitations, including: slow on/off times, difficultyin establishing well-defined spatial patterns of activation, andincreased potential for off-target effects. Similar concerns can beraised about the effects of heat shock or other broad environmentalchanges as activating stimuli.

In one embodiment, the light-responsive DNA-binding protein of theinvention comprises a light-responsive domain and DNA binding domainoperably linked or connected to a transcriptional activation domain. Incertain embodiments, the light-responsive domain and DNA binding domainare from a homologous species. In another embodiment, both domains maybe from the same species or may be encoded from the same gene or anaturally contiguous nucleic acid molecule.

Light-responsive domains comprise protein domains that function in alight dependent manner, for instance, by changing structure in responseto exposure to light. Light-responsive proteins and domains function inassociation with chromophores, which are moieties capable of detectingor capturing light energy. Any photosensory domain may be used as alight-responsive domain of the invention. In one embodiment thelight-responsive domain is a LOV domain. LOV domains are light sensitivedomains responsive to blue light that use a flavin mononucleotide, whichis widely available in eukaryotic cells, as a chromophore. The LOVdomain of the present invention may be any LOV domain known in the art.In one embodiment the LOV domain and the DNA binding domain of theinvention are from a homologous species and have, for instance,co-evolved to naturally function as a contiguous protein or nucleic acidmolecule. In a particular embodiment, the LOV domain of the presentinvention is a LOV domain from the E. litoralis 222 amino acid protein(EL222).

The EL222 protein consists of a photosensory (LOV) domain, aninterdomain linker (Jα-helix), and a helix-turn-helix (HTH) DNA bindingdomain (FIG. 1a ). In the dark, the LOV domain binds the HTH domain viainteractions of its β-sheet with the HTH 4α-helix. The HTH 4α-helixtypically provides a dimerization interface for DNA-bound HTH domains(FIG. 1a ). Illumination with blue light triggers a photochemicalreaction between the LOV domain and its flavin chromophore that leads toconformational changes that disrupt the LOV-HTH domain interactions andexpose the HTH 4α-helix. The HTH 4α-helix then binds to another HTH on asecond EL222 molecule generating an EL222 dimer that subsequently bindsDNA. In addition, the LOV domain photochemistry is reversible, in oneembodiment, spontaneously shutting itself off after about 30 seconds inthe dark.

In another embodiment, the system of the invention can be tuned to havedifferent kinetics of activation and/or inactivation using pointmutations. For instance, the rate of spontaneous “shutting off” can bealtered with point mutations, for instance, to residues near thechromophore binding site. Initial evaluation of several mutations asdescribed in the Examples below (Table 4) shows that these changes caneither accelerate this rate (e.g. Alanine 79 replaced by Arginine isaccelerated ten-fold) or slow it (e.g. a variant combining changes at41, 52, 79 and 121 exhibits 75-fold slower reversion). These variantscan be used individually or in combination with others in the table orother variants. Such variants find use in many techniques as will beappreciated by those in the art. For instance, in certain embodiments,such variants may be useful for applications where transiently-inducedgene expression is desired for studies of rhythmic biological phenomena.

In a further embodiment, the LOV domain may be activated or turned onagain after a period of inactivation by further illumination with bluelight. It is therefore possible to have multiple cycles of activationand inactivation through repeated cycles of blue light exposure anddark. In some embodiments, these pulses of light and dark may compriseequal periods of time in the light and time in the dark, or may compriseuneven periods, where the cells are exposed to light for a longer orshorter time than they are left in the dark. Such periods may comprise 5seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 60seconds, 70 seconds, 75 seconds, 2 minutes, 3, minutes, 4 minutes, 5minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 1hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9hours, 10 hours, 12 hours, 20 hours, 22 hours, 24 hours, 36 hours or 48hours. In other embodiments, the pulses may be repeated for multiplerounds of activation and inactivation, and such pulses may or may notcomprise equal periods of time per round.

DNA-binding domains, in general, are well known in the art, and referherein to protein domains that recognize a specific or consensus DNAsequence. Alternatively, a DNA binding domain may have a generalaffinity to DNA, without recognizing a specific sequence. The motif ormotifs within a DNA-binding domain that recognize DNA can recognize andbind to double- or single-stranded DNA. There are numerous DNA-bindingdomains known in the art, including helix-turn-helix domains,helix-loop-helix domains, zinc finger domains, leucine zipper domains,and high mobility group box domains. A DNA-binding domain of the presentinvention may comprise any DNA-binding domain known in the art.

In a particular embodiment, the light-responsive DNA-binding protein ofthe invention is operably linked to a heterologous regulatory orfunctional domain or protein. Such domains may comprise, for instance,general transcription factors, transcriptional activators,transcriptional enhancers and transcriptional repressors. In oneembodiment, the light-responsive DNA-binding protein of the presentinvention is operably linked to a transcriptional activation domain.Such linkage can be through the form of a fusion protein. A fusionprotein as referred to herein describes a protein operably linking orconnecting two or more proteins such that each protein continues toserve its intended function. Such proteins are typically linked viapeptide bonds and may be constructed using standard techniques known inthe art. It is understood that one of skill in the art may combinemultiple proteins to create fusion proteins and may also alter theproteins comprising the fusion protein by inserting, deleting orrearranging the amino acid sequence of the proteins or domains withinthe proteins to produce variants that retain the intended function. Alsoincluded herein are nucleic acids encoding a LRDP operably linked to atranscriptional activation domain.

The transcriptional activation domain may be selected from anytranscriptional activation domain known in the art, including but notlimited to, acidic transcriptional activation domains, such as from GAL4or the C-terminal portion of the herpes simplex virus viron protein 16(VP16); proline-rich transcriptional activation domains, such as fromCTF/NF1 or AP2; serine or threonine-rich transcriptional activationdomains, such as from ITF1 or ITF2; or glutamine-rich transcriptionalactivation domains, such as from Oct1 or Sp1. Other suitabletranscriptional activation domains are known in the art and would bereadily available to one of skill in the art.

The present invention is broadly applicable to gene expression in anycell, including eukaryotic cells, such as mammalian, insect, plant,yeast and fungal cells; and prokaryotic cells, such as bacterial cells.Cells encompassed by the present invention include individual cells,cell lines, cells in culture, cells to be modified for gene therapypurposes, cells modified to create transgenic or homologous recombinantorganisms, cells comprised in part of an organism or cells comprised inan entire organism. Examples of such cells include CHO dhfr-cells, 293cells, myeloma cells such as SP2 or NSO, hematopoeitic stem cells,myoblasts, hepatocytes, lymphocytes, neuronal cells, skin epithelialcells, airway epithelial cells, embryonic stem cells, fertilizedoocytes, plant root cells, leaf cells, flower cells, and cells fromseed. Accordingly, principles of the present invention include a cellcomprising a nucleic acid encoding a LRDP and/or a LRDP polypeptide.

Nucleic acids according to the invention, such as those encoding thelight-responsive DNA-binding domain protein or comprising DNA bindingsites may be introduced into a host cell for the regulation of geneexpression of such a cell in any manner known in the art, for instance,through transformation or transfection techniques known in the art.Techniques for transformation and transfection of animal, plant, fungal,insect and other cells are available to those of skill in the art andinclude, but are not limited to calcium phosphate co-precipitation,DEAE-dextran-mediated transfection, lipofection, electroporation,microinjection, polyethylene glycol-mediated transformation, viralinfection, Agrobacterium-mediated transformation, cell fusion, andballistic bombardment. Cells comprising the nucleic acids according tothe invention may be transiently or stably transformed. Such cells maytherefore transiently or stably express the gene product, such as theencoded light-responsive DNA binding domain of the present invention.Suitable methods for transforming host cells may be found in Sambrook,et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold SpringHarbor Laboratory press (1989)) and other laboratory manuals.

Regulatory sequences to be operably linked to nucleic acids according tothe invention for introduction into a host cell may include promoters,enhancers, leaders, introns, polyadenylation signals and otherexpression control elements. Regulatory sequences are known in the artand are available to those of skill in the art. (See, Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990)). The design of a suitable expression vector maydepend on various factors including the host cell to be transformed ortransfected or the level of gene expression desired. Nucleic acidmolecules according to the invention may therefore be introduced into ahost cell via a recombinant expression vector comprising such nucleicacids. Alternatively, nucleic acids according to the invention can beoperatively-linked to regulatory sequences, such as promoter, enhancer,leader or intron sequences, without additional vector sequences followedby introduction into a host cell.

The light-responsive DNA-binding protein regulates the expression of agene of interest comprising or operably linked to DNA-binding sitesrecognized by the DNA-binding protein. The light-responsive DNA-bindingprotein and the target nucleic acid sequence should therefore both bepresent in the host cell or organism. The present invention thereforeprovides cells comprising a nucleic acid molecule encoding thelight-responsive DNA-binding protein and/or a nucleic acid moleculecomprising a DNA-binding site recognized by the DNA-binding proteinoperably linked to a transcribable polynucleotide sequence.

The target nucleic acid sequence may comprise an exogenous orheterologous transcribable polynucleotide operably linked to aDNA-binding site transformed or transfected into the host cell ororganism in a manner similar to the introduction of the nucleic acidmolecule encoding the light-responsive DNA-binding protein, as describedabove. In such a case, the nucleic acid molecules encoding thelight-responsive DNA-binding protein and comprising the targettranscribable polynucleotide sequence can be introduced into a host cellor organism linked as a single molecule or as two separate molecules,for instance by co-transformation or successive transformation of onemolecule then the other.

Alternatively, the target nucleic acid molecule may be an endogenoustranscribable polynucleotide sequence. In such a case, the DNA-bindingsite is incorporated into the host cell in such a manner that it isoperably linked to the endogenous transcribable polynucleotide sequence.Operable linkage of the DNA-binding site to the endogenous transcribablepolynucleotide sequence may occur through any method known in the art,for instance, by homologous recombination between the two sequences.Homologous recombination techniques and methods are well known andavailable to one of skill in the art.

In certain embodiments, the transcribable polynucleotide sequencecomprised within the target sequence and that is operably linked to theEL222-binding consensus sequence may be any desirable transcribablepolynucleotide sequence. For instance, a reporter sequence, a cellstress tolerance sequence, an industrial enzyme encoding sequence; asequence encoding a biofuel production enzyme; a sequence encoding acell lysis protein or a sequence encoding a cell regulatory protein. Inparticular, embodiments, reporter sequences of the invention may be anyof the sequences known in the art that when expressed may be readilyidentified or measured. These sequences may be useful in identifying orselecting cells or individuals of interest and may include for instancegreen fluorescent protein (GFP) encoding sequences, luciferase encodingsequences, GUS genes, or lacZ genes. Cell stress tolerance sequenceuseful in the present application may provide tolerance to stressesincluding, but not limited to heat stress, drought stress, bioticstress, nutrient deficiency stress or oxidative stress. Many industrialenzyme encoding sequences are known in the art that may be useful in thepresent invention. These enzymes may include any enzyme that can providea functional use in an industrial or commercial setting, such asamylase, protease, trypsin, pectinase, lipase, lactase, xylanase orcatalase. One class of industrial enzyme may include biofuel productionenzymes such as cellulase or ligninase. Cell lysis proteins are alsowell known in the art and include any enzyme or other protein thatbreaks down the structural integrity of a cell, such as lysozyme,proteinase K or lysin. There are numerous sequences known in the artencoding cell regulatory proteins that may be useful in the presentinvention, such as kinase regulatory proteins, regulators of cellmetabolism, regulators of cell differentiation, or regulators of celldivision or growth.

The target nucleic acid sequence, in one embodiment may encode a proteinof interest for light-dependent control of the expression of suchprotein. Alternatively, the target nucleic acid sequence may betranscribed into an active RNA molecule, such as transfer or ribosomalRNA molecules, or into a RNA molecule for gene suppression in the cell,such as an antisense RNA, dsRNA, shRNA, siRNA or miRNA molecule.Transcription of such molecules under control of the system of thepresent invention may therefore provide light-dependent regulatorycontrol within the host cell, for instance, via protein expressioninhibition or suppression. In one embodiment, the system, methods,proteins and nucleic acids of present invention are therefore useful inany instance where it is desirable to control gene expression in atargeted, rapid and reversible manner without undesirable pleiotropiceffects or cytotoxicity. In a certain embodiment, the invention may beuseful, for instance, for developmental studies, in which geneexpression is only desired or necessary during a particular stage ofdevelopment; treatment of diseases via gene therapy, where localizedexpression is particularly desirable; removal or reduction ofundesirable gene products in a conditional manner via antisense orribozyme molecules, for instance to alter biochemical pathways; largescale production of a protein of interest when desired; production oftransgenic plants or animals without expression of the targettranscribable polynucleotide effecting proper development, or to targetparticular tissues within such transgenic organisms.

Expression of the target nucleic acid molecule is controlled viaexposure to blue light, for instance light at wavelengths about 350 nmto about 500 nm, for instance, about 400 nm to about 450 nm.

Variants of the light-responsive DNA-binding protein are also providedby the present invention. Nucleic acid and protein variations can bemade by one of skill in the art using conventional techniques well knownin the art. In one embodiment, variants of the light-responsiveDNA-binding protein alter the responsiveness and/or reversibility of theresulting gene expression. For instance, variants rendering thelight-responsive DNA-binding protein more or less responsive to the bluelight, or requiring more or less exposure to the light, or exposure formore or less time are encompassed by the present invention.

The invention also provides for methods for separation, isolation ordetection of at least one desired target moiety, such as a nucleic acidmolecule. Such methods comprise operably linking a DNA-binding siterecognizing a light-responsive DNA-binding protein of the presentinvention to the target moiety, contacting the light-responsiveDNA-binding protein of the present invention to the target moiety orsample comprising the target moiety in the presence of blue light andthus separating, isolating, or detecting the target moiety. Release andcollection for analysis such as identification of the target moiety maybe achieved by removing exposure to blue light. Alternatively, analysis,such as identification, may be performed while the target moiety isbound to the light-responsive DNA-binding protein. Detection moietiesmay also be used operably linked to the light-responsive DNA-bindingprotein. For instance light-responsive DNA-binding proteins bound in thepresence of blue light to DNA-binding sites operably linked to a targetmoiety may be distributed on an array and detected via an operablylinked detection moiety, such as a green florescent protein, greenflorescent protein variants, enzymatic label or other isotopic oroptically detectable labels.

Kits containing any components needed for light-regulated geneexpression are also provided in the present invention. In oneembodiment, a kit according to the present invention comprises a kit forthe separation, isolation or detection of at least one desired targetmoiety, such as a nucleic acid molecule. Such a kit may comprise nucleicacids of the invention, such as nucleic acids comprising the target DNAbinding sequences or proteins according to the present invention, suchas a light-responsive DNA-binding protein. A kit according to theinvention may further optionally comprise a column, an array, a bindingmatrix or bead, a detection moiety, such as an isotopic or opticallydetectable label, and any necessary buffers or solutions for thepurification or separation of the desired nucleic acid molecule.

In another embodiment, the present invention provides a kit useful forregulating expression a gene of interest in a target cell or organism.Such kits may include nucleic acids according to the invention, such asnucleic acids encoding a light-responsive DNA-binding protein or nucleicacid molecules comprising the target DNA binding sequences or both. Saidnucleic acid molecules may be provided or comprised within one or morevectors, such as an expression vector, and such vector may optionallycomprise further elements, such as an operatively-linked minimalpromoter, enhancer, intron or other regulatory sequence, or a cloningsite for the introduction of any genes of interest or genes targeted forexpression controlled via the light-responsive DNA-binding system of thepresent invention.

Alternatively, a kit of the invention may comprise cells comprisingnucleic acids according to the invention or cells in which nucleic acidsaccording to the invention have been stably incorporated therein. Suchnucleic acid molecules may comprise nucleic acids encoding alight-responsive DNA-binding protein or nucleic acid moleculescomprising the target DNA binding sequences or both. In one embodiment,a kit of the invention comprises cells expressing a light-responsiveDNA-binding protein of the invention. The present invention furtherprovides methods of using a kit of the invention.

In yet another embodiment, the present invention provides methods forselectively recruit proteins to a surface, such as the surface of asubstrate, for instance in a light-dependent manner. Such methods may inone embodiment be employed for use in a microarray system. Microarraysystems, such as those from Illumina or Affymetrix are well known in theart. In certain embodiments, such methods may comprise immobilizing toor coating a surface with DNA-binding sequence molecules, exposing thesurface to a light-responsive DNA-binding protein of the invention andselectively illuminating said surface. By DNA-binding sequence moleculesis meant nucleic acids having a sequence to which the DNA binding domainof an LRDP binds. Upon illumination, the light-responsive DNA-bindingprotein may become associated with DNA-binding sequence molecules on thesurface of the substrate, thus recruiting these proteins and any bindingpartner, such as a nucleic acid or protein, in association with orlinked to the light-responsive DNA-binding protein. Thus, in oneembodiment, the light-responsive DNA-binding protein may be associatedwith or linked to, for instance through a fusion protein, any protein ofinterest. In another embodiment, a light-responsive DNA-binding proteinof the invention may be recruited to a surface using such methods in aspatial pattern through selective spatial illumination of the surface.In some embodiments nucleic acids immobilized on the substrate surfacehave different sequences and are distributed at discrete sites on thesubstrate surface.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Engineering the EL222 Protein

The first step taken to adapt the EL222 prokaryotic transcription factorfor use in eukaryotic cells (FIG. 1) was to modify EL222 with nuclearlocalization and activation domains (not present in the naturalprotein), without hindering DNA binding or light regulation. Thetranscriptional activation domain (AD) from the herpes simplex virusVP16 protein, which is routinely used to efficiently recruit componentsof the mammalian transcription machinery to a target gene (Blau et al.,Mol Cell Biol (1996)16: 2044), was fused to the EL222 coding sequence(FIG. 1b ). A nuclear localization signal (NLS) was also added toproperly localize the VP16-EL222 fusion protein to the nucleus.Expression of the VP16-EL222 chimera is driven from the constitutivelyactive SV40 early promoter.

Methods

A. Vector Construction

DNA encoding a 13-residue N-terminal truncation of WT-EL222 (residues14-222), provided as SEQ ID NO:72, was cloned into the expression vectorpHis₆-Gβ1-Parallel1 (Nash et al., PNAS 108 (2011): 9449).

The rate-altering A79Q-EL222 variant, provided as SEQ ID NO:76, wasproduced using the QuikChange protocol (Stratagene) within the contextof the N-terminally truncated EL222 (Zoltowski et al., Biochemistry(2011): 8771). The A79Q-EL222 variant was subcloned into apHis₆-Parallel1 expression vector.

B. Purification of EL222 Protein

WT-EL222 and A79Q-EL222 proteins were expressed in E. coli BL21(DE3)cells. Cells were grown in LB-AMP at 37° C. in the dark and were inducedwith 0.5 mM IPTG. After a 20 hour induction at 18° C., cells werecentrifuged and resulting pellets resuspended in buffer A (50 mM Tris-ClpH 8.0 and 100 mM NaCl) and subsequently lysed by sonication. Theprotein was purified in the dark at 4° C. by gravity-flow chromatographywith Ni-NTA agarose (Qiagen) equilibrated in buffer A. Proteins wereeluted in buffer B (50 mM Tris-Cl pH 8.0, 100 mM NaCl, 75 mM imidazole),exchanged into buffer A and concentrated to 100-200 μM.

Example 2 Identification of High Affinity DNA Binding Sites

In addition to modifying the EL222 protein, a high-affinity cognate DNAbinding site (EL222) was identified and used to construct a reportervector with tandem copies of this and a minimal TATA-box promoter infront of a Firefly luciferase gene as a test transgene. Several siteswere initially identified for this purpose using a candidate-basedapproach based an in vitro DNA binding assay (EMSA, Electro MobilityShift Assay) of approximately 15 sequences located upstream of the EL222gene itself (Nash et al., 2011). From these candidates, one DNA site wasidentified (AN-45) that bound EL222 best in the group with an EC₅₀ of5-10 μM. Importantly, DNA binding occurred only when the protein/DNAmixture was exposed to light and not when kept in the dark, as expectedfrom the presence of the photosensitive LOV domain. However, while theAN-45 substrate bound with the highest affinity of this limited set ofoligonucleotides, it was suspected that this was not an optimal bindingsite given DNA-binding affinities for other HTH-containing proteins (Zhuand Winans, Proc. Natl. Acad. Sci. 98 (2001): 1507) and it did not proveto be useful in pilot studies of this light-activated gene system.

To identify additional DNA sequences with higher affinity for EL222, anin vitro selection method was used (SELEX, Systematic Evolution ofLigands by EXponential enrichment; Tuerk and Gold, Science 249 (1990):505). For this assay a large double-stranded oligonucleotide library(˜5×10¹⁴ molecules) was synthesized consisting of randomly generated 33bp sequences flanked by constant 5′ and 3′ ends (each 21 bp long) withprimer binding sites (FIG. 2a ). The oligonucleotide library was nextincubated with recombinant His₆-tagged EL222 protein in a 2:1(DNA:protein) ratio and the mixture was exposed to light to activate theprotein. The EL222/DNA complexes were purified by affinitychromatography using Ni-NTA beads under stringent binding conditions.Next, the purified EL222/DNA complexes were incubated in the dark toinactivate the protein and elute the DNAs. The DNA sequences wereamplified by PCR and then used to set up a second round of selection;the entire cycle was repeated a total of four times. Gel mobility shiftassays were done after each selection cycle to verify that the bindingaffinity of the DNA pools increased after each successive round (FIG. 2b, Rounds 1-4).

As predicted, by the fourth round of SELEX a pool enriched with DNAswith higher affinity for EL222 than the AN-45 substrate was obtained(FIG. 2b , Round 4 as compared to pre-selection). DNAs from SELEX round4 were further cloned and sequenced. A total of 60 DNA sequences werecloned, but because some of these sequences were repeated, only 57unique DNA sequences were obtained. A complete list of these sequencesis shown in Table 1 and Table 2. Based on analyses using the motif-basedsequence analysis tool MEME, seven DNA sequences were chosen forfollow-up binding studies (Table 1). EMSA assays showed that Clone-1bound with the highest affinity to EL222 out of the seven DNAs tested,with a 30-50 fold higher affinity than AN-45 (EC₅₀ value of 100-300 nM;Table 1, FIG. 2b ). To further delineate the sequence within 45-bpClone-1 DNA most relevant to EL222 binding, 4 overlapping 20-bpfragments derived from Clone-1 were designed and their binding to EL222was assessed by gel mobility shift assay (FIG. 3). Interestingly, onlythe C1-2 fragment, which contains the full motif identified by MEME (seeTable 1), was bound by EL222, while fragments C1-1 and C1-3 do not bindEL222 despite containing halves of the motif

TABLE 1Seven DNA sequences chosen for follow up and their half maximal effectiveconcentration (EC₅₀) values for binding to EL222. SEQ ID NameDNA sequence (33 bp randomized region) NO: EC₅₀(μM) Clone-1TTATAGGTAGCCTTTAGTCCATGCTGATTCGTT 4 0.1 < EC₅₀ < 0.3 μM Clone-3TAGACTTAAGTCTAGTACACAAGTTTTCCAGGG 5 0.3 ≦ EC₅₀ < 1 μM Clone-2AGCTAGGCTTTTGGTCTGTATAGCTGTCTTATA 6 0.5 < EC₅₀ < 1 μM Clone-22TATAGGGCTTTAGTCTGTATGTGGGATGTGTGT 7 0.5 < EC₅₀ < 1 μM Clone-53CATGGCCTAAGGACTGTAAGTACTATTAAATAC 8 0.5 < EC₅₀ < 1 μM Clone-77CAGATACATGCTTTAGTACGTATATTCGATGTC 9 0.5 < EC₅₀ < 1 μM Clone-78GCCCTAAGACTGTATCTTATTCGTGAGTTTTAA 10 0.5 < EC₅₀ < 1 μMConsensus motif: G[C/G]CTT[T/A][A/G]G[T/A]C[T/C]GTA 67 The identified 14bp consensus motif is shown in bold.

TABLE 2 List of 50 additional DNA sequences derived from SELEX. SEQ EC₅₀ID Name DNA sequence (μM) NO: Clone-4 AAATCGAGGGGCCGAGGTCTCCTTTCCTTGACAn.d. 11 Clone-5 ACCTTACAAAGCTAATAATTCTTCCTTCAAACG n.d. 12 Clone-6TGTTCAGGGGTATGCCTACGCTGGATCAGCCGT n.d. 13 Clone-9GTGACCGTTCCGTATTCATTATTCACACATAAT n.d. 14 Clone-10TCGCATTAGAATGGCTGGGACGGTTAGTGTTGG n.d. 15 Clone-15TTAGGCACTTGTGACTTGACGTTGTTTGTACAT n.d. 16 Clone-16TTCTGAGGAAGATGAAGATGACAGGCATGTGTA n.d. 17 Clone-17CAGTCTTTCTCTCTCGTCGACGATGTATTTTCT n.d. 18 Clone-18ATTTTTGGTGGGAGTATTAGTACGAACGGCTGT n.d. 19 Clone-29TTCAATCGCTAGCTACCTTACTCGTTAATAAAG n.d. 20 Clone-38GACCAAAGTGCAATGGTCCAATTCGGTTATGCC n.d. 21 Clone-39GTATTTAATTGCAGAGAAAATCTTTATCTTCTT n.d. 22 Clone-40GTTCGCTTTGAATGTAACGGACTCGTGAGATTT n.d. 23 Clone-41CATTCAAAAGTACGCGAGGTTTTGGTGCTACTT n.d. 24 Clone-42GTCTAAGGACTAGGTGTGTTCCTCTGGATCTCG n.d. 25 Clone-43AGGGTCGCTCAGCATAAAGGTTGCAATTACGGT n.d. 26 Clone-44AAAGTCTAGTATTGTACACCTCCTCTCGGTAAA n.d. 27 Clone-45ATCAGGGACATAGAACGCTAAGACTCGCCTGCA n.d. 28 Clone-46GGTGCCGGTGAGTACCATGGACCTTGGTAACTT n.d. 29 Clone-47AAGTGACGATTACATACCCCGTATACCGTATCA n.d. 30 Clone-48CTGATACTATAGCTACCTTTGATGGTTTTCATA n.d. 31 Clone-49GTCTTTTGGGCATGTTGTGAATAAGAAGCCAAA n.d. 32 Clone-50ACAAAAGCCTGTGCGGTAAGAAATGAGTTTTAA n.d. 33 Clone-54ACCCAGTCGACTTCAAGGATATAATAAACCCGT n.d. 34 Clone-55TACTTAAGGACTATATAAGGGTTTACTGATCGT n.d. 35 Clone-56TTGGTACATTATACAAGCGTGAGAGGACTGGCT n.d. 36 Clone-57TCGAAAAGCTACGGTCATTATGTAGGACTGTCG n.d. 37 Clone-61TACGTTGGGTGTTTTTTAATCCCGGAAATGCGT n.d. 38 Clone-62CAGTCTTTATCTCTCGTCGACGATGTATTTTCT n.d. 39 Clone-63CGTCGATGAGAACCTCGGAGACATGGCCAGTCT n.d. 40 Clone-64CTATGTATCTACGTGAATGGATACGGTCTTTTT n.d. 41 Clone-65TATTAAGCCTAGGGGTCCTATTACCCTTAGACT n.d. 42 Clone-66CTATAGGTGACTGCGGGTATGAGAGTAGTGGGA n.d. 43 Clone-67TAGCCCATTTATTGTTTGAAAGCGCATTTGTCA n.d. 44 Clone-68GTTATGTTTGGCGACGCGCCGGTTAGTCTCCTT n.d. 45 Clone-71GCCTTTATGCTTATACGTGCCGATTTCCCAAAT n.d. 46 Clone-74TTATTAAGTCCGTATTAAGTATGGAGGGAGAGG n.d. 47 Clone-76GCACAATGTCCACCCTCACGCCATACTTATTGA n.d. 48 Clone-79GTTCCTTGGGATGAGCTCGTATGCACATGGTAT n.d. 49 Clone-81GGTTGAATAGTTATAATGCGACCTGGACTCTTT n.d. 50 Clone-82CAAACGTCATGGTCAACGTATTTACACTAGATC n.d. Si Clone-83TGTTGAGTTCATAGGAAGGGGCTGTAGACATTC n.d. 52 Clone-84CCATTTGAGATGTATAGCGGACCAGAAATGGTT n.d. 53 Clone-85CGATTGACTGTATGGACCTTAATATAGTTTGTA n.d. 54 Clone-86TACTATAGGATTGTTAGCCCTGAAGAGCAGGTG n.d. 55 Clone-87CAGTGCGCTAGCTAATACTCCCTTGTTTTACCT n.d. 56 Clone-88ACCTTATGACCAGGTTGTTGGACTGATTGTAAC n.d. 57 Clone-89TCCTTCAGTCTCTACTTATTAAAGGCTTGAAAG n.d. 58 Clone-90TCAGGGGACATTGGGCTACGATTATCTACCTTA n.d. 59 Clone-91GTCGTTAGGAGGTACTTTGAACGCCACCATCAT n.d. 60 Clone-92CCTGCTTGGCAAGATCTTACCGGGGTTTGGTAG n.d. 61 Clone-93GTATTTAAGCCATACTATTGTCTTAGCCGCGAG n.d. 62 n.d. - not determined.

TABLE 3 List of the DNA sequences derived from Clone-1,used to map regions of specific binding (FIG. 3).Each sequence is 20 bp long. SEQ ID Name DNA sequence EC₅₀ (μM) NO. C1-1TCTACGTTATAGGTAGCCTT n.d. 63 C1-2 TAGGTAGCCTTTAGTCCATG 0.3 < EC₅₀ < 0.564 μM C1-3 TTTAGTCCATGCTGATTCGT n.d. 65 C1-4 TGCTGATTCGTTTTCAACTT n.d.66 n.d. - not determined.

TABLE 4 Rate-altering variants of EL222 and their effect on EL222kinetics. EL222 Variant Lit state lifetime @ 25° C. SEQ ID NO: wildtype 25 s 73 V41I >25 s 74 L52I >25 s 75 A79Q 300 s 76 A79R  2.5 s 77 A79T 8 s 78 V121I >25 s 79 V41I:L52I >25 s 80 V41I:A79Q >25 s 81 V41I:V121I300 s 82 L52I:A79Q >25 s 83 V41I:L52I:V121I 1800 s  84V41I:L52I:A79Q:V121I 1800 s  85

Lit state lifetimes were measured by visible absorbance spectroscopyafter excitation. Values designated “>25 s” are approximate.

Methods

A. SELEX Procedure

The initial single-stranded oligonucleotide5′-GGGAATGGATCCACATCTACG-(N)₃₃-TTCAACTTGACGAAGCTTGCC-3′ (SEQ ID NO:1)was chemically synthesized by Integrated DNA Technologies (IDT). Toamplify the DNA pool, six 50 μl PCR reactions were set up using (finalconcentrations): 0.1 μM of the synthetic pool oligonucleotide astemplate, 2 μM primers, 200 μM dNTPs, 20 mM Tris-Cl pH 8.8, 10 mM(NH₄)₂SO₄, 10 mM KCl, 2 mM MgSO₄, 0.1% Triton X-100, 4 mM MgCl₂, and0.04 U Vent (New England Biolabs Inc.). The primers used were Fwd-L15′-GGGAATGGATCCACATCTACG-3′ (SEQ ID NO:2) and Rev-L15′-GGCAAGCTTCGTCAAGTTGAA-3′(SEQ ID NO:3). The cycling parameters were asfollows:

Initial denaturation: 94° C.  2 min 11 cycles: 94° C. 45 sec 60° C. 45sec 72° C. 45 sec Final extension: 72° C.  5 min

Amplified DNAs were purified using QIAquick PCR purification kit(Qiagen). Approximately 15.7 μM DNA (5×10¹⁴ molecules) and 7.8 μMA79Q-EL222 protein were incubated in a total volume of 500 μl containing(final concentrations): 10 mM Tris-Cl pH 8.0, 80 mM NaCl, 3 mM MgCl₂,10% glycerol, 0.025 mg/mL polydI-dC, and 0.01 mg/mL BSA. The bindingreaction was mixed by rotation for 25 min at 4° C. and kept undercontinuous illumination with a white LED light. Ni-NTA agarose beads(Qiagen) were pre-blocked with 0.02 mg/mL of polydI-dC and then added tothe binding reaction and incubated for an additional 25 min at 4° C.with mixing and continuous illumination. The bead/EL222/DNA complexeswere pulled down by centrifugation at 2000 rpm for 1 min at 4° C. Next,complexes were washed with 300 μl of buffer C (50 mM Tris-Cl pH 8.0, 300mM NaCl) and pulled by centrifugation, this step was repeated at least 2more times. After the last wash, the bead/EL222/DNA complexes wereresuspended in 400 μl of binding buffer (without polydI-dC and BSA) andincubated in the dark for 30 min at 4° C. with mixing by rotation. Thebeads/EL222 complexes were pulled down by centrifugation and thesupernatant (containing the DNAs) was transferred to a new Eppendorftube.

Phenol/chloroform/isoamyl alcohol was added to the supernantant in a 1:1ratio, the sample was vortexed and centrifuged at 13,000 rpm for 5 minat 4° C. The top aqueous phase, which contains the eluted DNA, wastransferred to a new Eppendorf tube containing 1 ml of 100% ethanol, 0.3M NaOAc pH 5.2, and 0.01 mg/mL glycogen. The DNA was precipitatedovernight at −20° C. and subsequently recovered by centrifugation at13,000 rpm for 20 min at 4° C. The pelleted DNA was resuspended in 12 μlof DNase-free water and later used as the template DNA pool in a secondPCR amplification step and round of selection. To sequence individualDNA sequences from the DNA pools obtained after each SELEX round, theDNA pools were cloned into the pBlueSkript+ vector using BamHI andHindIII restriction sites.

B. Electrophoretic Mobility Shift Assay

DNA pools derived from each SELEX cycle were PCR amplified as describedpreviously in the SELEX procedure. Complementary oligonucleotides foreach individual SELEX-derived clone sequence were chemically synthesized(Sigma), heated to 95-100° C. for 5 min and left to cool to roomtemperature to anneal the oligonucleotides. DNAs were 5′-end labeled ina 50 μl reaction containing (final concentrations) 68 nM DNA substrate,70 mM Tris-Cl pH 7.6, 10 mM MgCl₂, 5 mM DTT, 0.6 μCi ATP [γ-³²P] (PerkinElmer, cat. no. BLU502Z500UC), and 0.2 U PNK (New England Biolabs Inc.).The reaction was incubated for 30 min at 37° C., followed by a 20 minincubation at 65° C. to heat inactivate the enzyme. The ³²P-labeled DNAwas purified from the unincorporated ATP [γ-³²P] using IllustraProbeQuant G-50 micro-columns (GE Healthcare). Approximately 13.6 nMradiolabeled DNA was incubated with varying concentrations of WT-EL222in the same binding buffer used for SELEX procedure for 25 min at 4° C.with continuous illumination with a white LED light. Reactions wereanalyzed on a 5% native gel (Acrylamide/Bis 29:1) and run in TBE bufferat 150 V for 1.5 hr at 4° C. The gel was exposed to a PhosphorImagingplate and visualized using FujiFilm FLA-5100 imaging system.

Example 3 Light-Dependent Gene Activation in Eukaryotic Cells

Based on the in vitro binding data in Example 2, two reporter constructswere designed to test in cultured mammalian cells together with theVP16-EL222 fusion protein. Both constructs contained the Fireflyluciferase gene under the control of five tandem copies of either theClone-1 45-bp sequence or the C1-2 20-bp sequence (FIG. 4a ). Whenco-transfected into 293T cells, the VP16-EL222 expression construct wasable to activate transcription of the pClone1-45[Luc] andpClone1-20[Luc] reporters by 57-fold and 87-fold over empty vectorcontrols respectively, when the cells were exposed to blue light (FIG.4b ). In contrast, dark state controls only showed about a 1.5 to 2-foldchange in luciferase induction over empty vector for both reporters.After correcting for this small background induction in the dark, wecalculated that for pClone1-45[Luc] and pClone1-20[Luc] there is 25-foldand 58-fold increase in transgene expression dark-to-light,respectively. With changes to the illumination protocol to optimizeconditions given the lifetime of the wildtype EL222 photoexcited state,we observed even higher fold activation (e.g. 108-fold activation, FIG.5). Additional control experiments have shown that the activation ofVP16-EL222 in mammalian cells is specific to blue light, as illuminationwith red light does not turn on transcription of Luciferase reporter(FIG. 7a ). The specificity of the VP16-EL222 protein for the 20 bp C2-1sequence (SEQ ID NO: 69) when transfected into mammalian cells wasverified by experiments wherein reporter vectors under the control ofnonspecific DNAs sequences were shown to not be turned on by EL222either in the dark or with light (FIG. 7b ). Taken together, this datademonstrate that the EL222 can function as a blue-light-regulatedtranscription factor that can be used to control transgene expression inmammalian cells.

Methods

A. Mammalian Vector Construction

For expression in mammalian cells, DNA containing the 13-residueN-terminal truncation (residues 14-222) of WT-EL222 was subcloned intothe mammalian expression vector pVP16 (Clontech; catalog no. 630305)using EcoRI and XbaI restriction sites to obtain pVP16-EL222. TheVP16-NLS-EL222 coding sequence is provided as SEQ ID NO:70. Thecorresponding amino acid sequence is provided as SEQ ID NO:71. Thenuclear localization signal (NLS) corresponds to nucleotides 7-27 of SEQID NO:70 and amino acids 3-9 of SEQ ID NO:71. The VP16 activation domaincorresponds to nucleotides 28-261 of SEQ ID NO:70 and amino acids 10-87of SEQ ID NO:71. The EL222 ORF (14-222 aa) sequence corresponds tonucleotides 268-879 of SEQ ID NO:70 and amino acids 90-298 of SEQ IDNO:71 (FIG. 6). The NLS and VP16 regions were derived from the pVP16Clontech vector. The rate-altering variants L52I-EL222 (SEQ ID NO:75),A79Q-EL222 (SEQ ID NO:76), and L52I/A9Q-EL2222 (SEQ ID NO:83) weresubcloned into the pVP16 vector from Clontech (see FIG. 10).

The five tandem copies of the Clone-1 sequence (both the 20 bp and 45 bpversion as shown in FIG. 4) were chemically synthesized by GeneArt(Invitrogen) and inserted into the pGL4.23[luc/minP] construct (Promega)using XhoI and HindIII restriction sites to obtain pClone1-20[FireflyLuc] or pClone1-45[Firefly Luc]. To make the mCherry reporter constructthe Firefly Luciferase coding sequence in pClone1-20[Firefly Luc] wasreplaced by the mCherry coding sequence using NcoI and XbaI. Theresulting vector was named pClone1-20[mCherry]. Additional vectors weremade that served as controls in experiments, these were thep3XAN45[Firefly Luc] and p5XUAS_(G)[Firefly Luc] constructs. Thep3XAN45[Firefly Luc] vector was made by inserting three tandem copies ofthe AN45 DNA sequence (Nash et al., PNAS 108 (2011): 9449), which werechemically synthesized by GeneArt (Invitrogen), into thepGL4.23[luc/minP] construct (Promega) using XhoI and HindIII. Similarly,for the p5XUAS_(G)[Firefly Luc] vector five tandem copies of the GAL4upstream activation sequence (UAS_(G)) were amplified from the pG5SEAPvector (Clontech; catalog no. 630305) and subcloned into thepGL4.23[luc/minP] using XhoI and HindIII.

B. Light-Activation of Luciferase Transcription

i. Constant Illumination

Human embryonic kidney cells (HEK-293T cells; ATCC) were cultured inDMEM (Thermo Scientific; cat. no. SH30284.01) supplemented with 10%fetal calf serum (FCS) and 1% penicillin/streptomycin (Pen/Strep)solution at 37° C. in 5% CO₂. Cells were co-transfected usingLipofectamine 2000 (Invitrogen) in an optimized protocol. On the day oftransfection, a total of 495 ng of DNA (410 ng of pVP16(empty) orpVP16-EL222 DNA, 82 ng of pClone1-20[Firefly Luc] or pClone1-45[FireflyLuc] DNA, and 3 ng of pGL4.75 [hRluc/CMV] (Promega)) were diluted in 50μl of Opti-MEM I Medium (Invitrogen; cat. No. 11058) and aliquoted intoa 24-well plate. For each well, 1.25 μl of Lipofectamine 2000 wasdiluted in 50 μl of Opti-MEM I Medium and incubated for 5 min at roomtemperature. The diluted Lipofectamine 2000 was then added to each wellcontaining the diluted DNA and incubated at room temperature for 20-30min. During this incubation step the cells were trypsinized, washed with10 mL of phosphate buffered saline solution and centrifuged at 1000 rpmfor 5 min at room temperature. The cells were then resuspended inPen/Strep-free DMEM+10% FCS and diluted to 1×10⁵ cells per 500 μl. Afterthe 30 min incubation step, 500 μl of the cell suspension were added toeach of the wells containing the diluted DNA/Lipofectamine 2000 mixture.At 24 hr after transfection, a blue LED array (LED wholesalers; 2501BUblue 225 LED 13.8 Watt square panel, 110 V) was placed above the‘light-treated’ plate inside the cell incubator for an entire 24 hrperiod. The non-treated plate was kept in the dark throughout theexperiment. At 48 hr after transfection, Luciferase activity wasmeasured with the Dual-Glo Luciferase Assay system according to themanufacturer's instructions (Promega). A single experiment includedidentical transfections set up in triplicate for each condition (i.e.,pVP16 (empty) and pVP16-EL222). The Firefly Luciferase data wasnormalized relative to Renilla Luciferase to correct for differences intransfection efficiency between samples. For this, the average of thetriplicate Firefly Luciferase values are divided by the average of thetriplicate Renilla Luciferase values for each condition. Next a ratio ofratio is calculated to obtain the fold change (FC) in activation,FC=[(Firefly/Renilla)_(VP16-EL222)/(Firefly/Renilla)_(empty vector)]. Tocalculate the fold change upon blue-light illumination, the FC_(Light)for pVP16-EL222 was divided by the FC_(Dark) for pVP16-EL222.

ii. Optimized Illumination Based on the Lifetime of EL222 PhotoexcitedState

A Firefly luciferase reporter construct under the control of 5 copies of20-bp C1-2 (SEQ ID NO:69) sequence was co-transfected with theVP16-EL222 expression construct or a VP16(empty) vector into HEK293Tcells in a manner similar to that above. At 24 hr after transfection,the cells were kept in the dark or illuminated with blue light in analternative fashion for 24 hours using an illumination schedule of (20seconds light:60 seconds dark) through the 24 hour period. Afterwards,cells were harvested and luciferase activity was measured (left graph).Luciferase values were normalized for transfection variability using acontrol Renilla luciferase reporter with a constitutively active CMVpromoter and was co-transfected with the Firefly luciferase reporter andVP16-EL222 expression vector. Results are provided in FIG. 5. A 108-foldenhancement in gene expression was observed with this illuminationprotocol, compared to 58-fold change observed with the same enhancer andconstant illumination (FIG. 4b ).

iii. Stable Cell Line that Expresses VP16-EL222 Protein

DNA containing the VP16 activation domain, the NLS, and the 13-residueN-terminal truncation (residues 14-222) of WT-EL222 was PCR amplifiedfrom the pVP16-NLS-EL222 vector and subcloned into the mammalianexpression vector pIRESpuro (Clontech; catalog no. 6031-1), whichcontains a Puromycin resistance gene, using EcoRV and BamHI. Theconstruct was named pIRESpuro-VP16-EL222. HEK293T cells were transfectedwith the pIRESpuro-VP16-EL222 and at 24-hr after transfection the cellswere cultured in media containing puromycin at 2 μg/ml to select forthose cells carrying the pIRESpuro-VP16-EL222 vector.Puromycin-resistant colonies were validated for expression of VP16-EL222protein by western blotting with an anti-VP16 antibody (Abcam; catalogno. ab4808) (see section “vi” below). Of the colonies screened, theVP16-EL222 stable cell line named Clone-3 showed the highest level ofprotein expression and was therefore chosen for use in subsequentexperiments.

The protocol for Luciferase transcription assays in the VP16-EL222stable cell line was as follows. Clone-3 cells were transfected with theFirefly luciferase reporter construct under the control of 5 copies of20-bp C1-2 sequence (SEQ ID NO:69) only. Immediately after transfectionthe cells illuminated with blue light in an alternative fashion for 3,6, 9, 12 or 24 hours using an illumination schedule of (20 secondslight:60 seconds dark) or kept in the dark for the duration of theexperiment. After each time point the cells were harvested andluciferase activity was measured. Results are provided in FIG. 11.Luciferase intensity was measured at over 1.3 million relative lightunits (RLU) in Clone-3 cells after 12 hr of illumination. Compared tothe Luciferase intensity values we obtained when HEK293T cells weretransiently transfected with VP16-EL222 and the p5XClone1-20[FireflyLuc] reporter vectors (˜50-60,0000 RLUs), we observed over 20-foldhigher light-driven activation of Luciferase transcription with theClone-3 cell line that stably expresses VP16-EL222 (compare FIG. 5 (leftpanel) and FIG. 11a ).

The results in FIG. 11a show that the highest level of Luciferasetranscriptional activation in Clone-3 cells was achieved after 12 hr ofillumination. With this information in hand the parameters of theillumination schedule were tested. Three different illumination cycleswere compared: 20 seconds light/60 seconds dark; 10 seconds light/70seconds dark; and 5 seconds light/60 seconds dark. The results (shown inFIG. 11b ) demonstrate that the optimal illumination schedule consistsof 20 seconds light/60 seconds dark cycle for a 12 hr period.

iv. Cell Viability Assay

The CellTiter-Blue® cell viability assay (Promega; catalog no. G8080)was used, as per manufacturer's instructions, to measure cell viabilityafter performing a constant illumination experiment as described abovein section called “i. constant illumination.”

v. Imaging of HEK293T Cells

An mCherry reporter construct under the control of 5 copies of 20-bpC1-2 (SEQ ID NO:69) sequence was co-transfected with the VP16-EL222expression construct or a VP16(empty) vector into HEK293T cells asdescribed above. At 24 hr after transfection, the cells were kept in thedark or illuminated with blue light in an alternative fashion for 24hours using an illumination schedule of (20 seconds light:60 secondsdark). Next, the cells were analyzed using a Nikon Eclipse TS100fluorescence microscope and images captured using NIS-Elements imagingsoftware. A surface plot representation of each of the images taken wasobtained using ImageJ software. The results are provided in FIG. 9.Cells expressing VP16-EL222 and that were illuminated with blue lightshowed high levels of mCherry fluorescence, indicative oftranscriptional activation by VP16-EL222. However, cells transfectedwith VP16-EL222 that were kept in the dark showed little to no mCherryfluorescence.

Similar experiments were done with the Clone-3 cell line that stablyexpresses the VP16-EL222 protein. In this case, Clone-3 cells weretransfected with the pClone1-20[mCherry] vector only and subsequentlyilluminated with blue light pulses (20 seconds light/60 seconds dark)for 24 hr. Cells were then analyzed using a Nikon Eclipse TS100fluorescence microscope as described above. The number of mCherrypositive cells was higher and the intensity of the fluorescent higher inClone-3 cells than in HEK293T cells that were transiently transfectedwith VP16-EL222 (compare FIG. 9 and FIG. 12). These data together withthe results presented in FIG. 11 suggest there is a notable advantage tousing a cell line that stably expresses the VP16-EL222 protein.

vi. Western Blotting to Determine Luciferase Protein Levels in HEK293TCells that Stably Express VP16-EL222

Clone-3 cells were plated at a density of 2.2 million cells/mL in atotal volume of 2 mL in a 6-well plate. The next day cells weretransfected with the Luciferase reporter vector containing 5 copies of20-bp C1-2 (SEQ ID NO:69) sequence as described above. Immediately aftertransfections cells were either kept in the dark or illuminated withblue light pulses using a 10 seconds light/70 seconds dark cycle for 12hr. Next cell lysates were made from dark and illuminated samples,approximately 10-20 μg of protein was loaded on a SDS-PAGE gel andresolved by electrophoresis. The proteins resolved from the gel weretransferred to a PVDF membrane (GE Healthcare) and blotted with ananti-VP16 antibody (Abcam; ab4808). The results are provided in FIG. 11c. These data show that Luciferase protein is expressed exclusively whenVP16-EL222 is present in cells and only when those cells are illuminatedwith blue light.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and in the steps or in the sequence of steps of the methoddescribed herein without departing from the concept, spirit and scope ofthe invention. More specifically, it will be apparent that certainagents which are both chemically and physiologically related may besubstituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

1.-40. (canceled)
 41. A recombinant nucleic acid molecule comprising apolynucleotide sequence encoding a light-responsive DNA protein (LRDP)comprising (a) a light-oxygen-voltage (LOV) domain, and (b) aDNA-binding domain (DBD), wherein the LOV domain and DBD are derivedfrom Erythrobacter litoralis 222 amino acid protein (EL222); wherein thesequence encoding the LRDP is operably linked to a polynucleotideencoding a heterologous transcription activation domain; and wherein thesequence encoding the LRDP comprises a point mutation selected from thegroup consisting of V41I, L52I, A79Q, A79R, A79T and V121I.
 42. Therecombinant nucleic acid molecule of claim 41, wherein the pointmutation is V41I.
 43. The recombinant nucleic acid molecule of claim 41,wherein the point mutation is L52I.
 44. The recombinant nucleic acidmolecule of claim 41, wherein the point mutation is A79Q
 45. Therecombinant nucleic acid molecule of claim 41, wherein the pointmutation is A79R.
 46. The recombinant nucleic acid molecule of claim 41,wherein the point mutation is A79T.
 47. The recombinant nucleic acidmolecule of claim 41, wherein the point mutation is V121I.